SUMO proteins are a family of ubiquitin-related proteins that become covalently linked to other cellular proteins. While budding yeast has a single SUMO, called Smt3p, there are three commonly expressed mammalian SUMO paralogues, called SUMO-1, -2 and -3. SUMO-2 and -3, are 96% identical, while SUMO-1 is roughly 45% identical to either SUMO-2 or -3. Human SUMO paralogues have been implicated in a variety of cell functions, including nuclear trafficking, chromosome segregation, chromatin organization, transcription and RNA metabolism. The conjugation pathway for SUMO proteins is similar to the ubiquitin conjugation pathway: SUMO proteins are processed by Ubiquitin like proteases/Sentrin specific proteases (Ulps/SENPs) to reveal a di-glycine motif at their C-termini. After processing, SUMO proteins undergo ATP-dependent formation of a thioester bond to their activating (E1) enzyme, Aos1/Uba2. The activated SUMO proteins are transferred to form a thioester linkage with their conjugating (E2) enzyme, Ubc9. Finally, an isopeptide bond is formed between SUMO proteins and substrates through the cooperative action of Ubc9 and protein ligases (E3). The linkage of SUMO proteins to their substrates can be severed by SUMO proteases, so it is likely that SUMO modification is highly dynamic in vivo. Like processing, SUMO deconjugation is mediated by Ulps/SENPs. [unreadable] To assess the differences between the SUMO paralogues, we have previously examined the behavior of YFP-SUMO fusion proteins that were stably expressed in HeLa cells. YFP-SUMO-1 behaved in a manner that is clearly distinct from YFP-SUMO-2 and YFP-SUMO-3: As described for endogenous SUMO proteins, YFP-SUMO-1 showed distinct responses to physiological stimuli, such as heat stress. YFP-SUMO-1 distribution overlapped with YFP-SUMO-2 and -3 in the nucleoplasm and in PML bodies, enigmatic nuclear structures that contain the promyelocytic leukemia gene product (PML). However, YFP-SUMO-1 was uniquely localized to nucleoli, the nuclear envelope and cytoplasmic foci. Finally, YFP-SUMO-1 was less dynamic than YFP-SUMO-2 or -3, with slower rates of both recovery in fluorescence recovery after photobleaching (FRAP) experiments and slower depletion during fluorescence loss in photobleaching (FLIP) experiments. This was true even when apparently indistinguishable nucleoplasmic regions were bleached, suggesting that YFP-SUMO-1s distinct dynamics are not a result of its sequestration in particular subnuclear regions. In combination with differences in overall abundance, profiles of conjugation targets, and chain-forming properties, these results argue that vertebrate SUMO paralogues are functionally distinct, although their individual roles remain to be fully understood.[unreadable] Ulp/SENPs play an important role in determination of the spectrum of conjugated species because they directly regulate the production of free, conjugatable SUMO proteins and the half-life of conjugated species. Moreover, yeast Ulp/SENPs are both cell cycle regulated and required for mitotic events, particularly chromosome segregation. S. cerevisiae has two SUMO-specific proteases, Ulp1p and Ulp2p. Ulp1p is encoded by an essential gene, and it localizes to nuclear pore complexes (NPCs). NPC targeting of Ulp1p is not required for its enzymatic function nor for rescue of cells lacking Ulp1p, but this distribution controls its access to potential substrates. Genetic analysis suggests that Ulp1p has an important role in processing of the single SUMO family protein in yeast, Smt3p. Ulp2p is a nucleoplasmic protein that is dispensable for growth. Ulp2p is important for deconjugation, and particularly for removal of poly-Smt3p chains. The situation is more complex in vertebrates, because there are multiple SUMO paralogues and a larger number of Ulp/SENPs. Humans have six Ulp/SENPs (SENP1, 2, 3, 5, 6 and 7), and Xenopus have five (xUlp1, SENP3, 5, 6 and 7). Among the mammalian SENPs, SENP1, 2, 3, and 5 are most closely related to Ulp1p, while SENP6 and 7 are more closely related to Ulp2p. We wish to determine the mitotic roles of Ulp/SENPs in vertebrate systems, and to assess which vertebrate enzymes play roles equivalent to the yeast proteins, if any. As a prelude to such experiments, we defined the evolutionary relationships among Ulp/SENPs, as well as their localization and enzymatic specificities. For the latter, we cloned cDNAs encoding each of the human and Xenopus laevis SENP proteins and raised antibodies against them.[unreadable] To assay the paralogue specificity of mammalian SENP/Ulps under comparable conditions, we collaborated with Keith Wilkinson (Emory University) to examine their reactivity with HA-tagged SUMO-1 and SUMO-2-vinyl sulfones (HA-SUMO1-VS and HA-SUMO2-VS). These reagents covalently react with active site cysteine residues of SUMO proteases in a manner that reflects the selectivity of the enzymes to individual paralogues. GFP fusions with all SENPs reacted with HA-SUMO2-VS, but only GFP-SENP1 and GFP-SENP2 showed substantial reactivity with HA-SUMO1-VS. Notably, the reaction of HA-SUMO1-VS with GFP-SENP1 was more efficient with GFP-SENP2, consistent with earlier observations of the binding of these proteases to individual paralogues. In conjunction with earlier reports, our findings indicate that SENP1 and SENP2 are the primary enzymes responsible for SUMO-1 metabolism, while all SENP/Ulps act on SUMO-2 and -3.[unreadable] We are systematically examining the localization and function of individual Ulp/SENPs. SENP6 (also called SUSP1) localizes within the nucleoplasm. SENP6 depletion from cell lines expressing GFP fusions to individual SUMO paralogues caused re-distribution of GFP-SUMO-2 and -3 into PML bodies, as well as PML body enlargement and increased PML body number. We did not observe comparable re-distribution of GFP-SUMO-1. While SENP6 has a strong paralogue preference for HA-SUMO2-VS, we were surprised to find that it had little activity for SUMO-2 or -3 processing or for the deconjugation of substrates containing fewer that three SUMO-2/3 moieties. By contrast, it was highly active against model substrates containing chains of SUMO-2 that were produced in a bacterial expression system. Together, our findings argue that SENP6 may play a specialized role in dismantling highly conjugated SUMO-2 and -3 species. This finding is particularly interesting in light of the established role of Ulp2p in chain editing in yeast. The other vertebrate Ulp2p-like enzyme, SENP7, likewise a nucleoplasmic SUMO-2/3-specific isopeptidease, although we have not yet determined whether it acts preferentially on chains. [unreadable] We have examined which Ulp1-related mammalian Ulp/SENPs localize to NPCs. Earlier, we and others showed that SENP2 binds the nucleoplasmic side of the NPC. Various localization patterns for SENP1 fusion proteins have been reported. Using specific anti-SENP1 antibodies, we found that endogenous SENP1 localized to inner face of NPCs in HeLa cells. The differences between our data and previous reports may reflect overexpression of SENP1 fusion proteins: We found that SENP1-NPC association occurred through saturable binding sites. The localizations of SENP1 and SENP2 indicate that both enzymes capable of efficient SUMO-1 deconjugation are restricted to NPCs, and may suggest that SUMO-1 has a role that is associated with or regulated by NPCs.